The mechanisms that ensure the removal of damaged mitochondrial proteins and lipids are critical for the health of the cell, and errors in these pathways are implicated in numerous degenerative diseases. We recently uncovered a new pathway for the selective removal of proteins mediated by mitochondrial derived vesicular carriers (MDVs) that transit to the lysosome. However, it was not determined whether these vesicles were selectively enriched for oxidized, or damaged proteins, and the extent to which the complexes of the electron transport chain and the mtDNA-containing nucloids may have been incorporated. In this study, we have developed a cell-free mitochondrial budding reaction in vitro in order to better dissect the pathway. Our data confirm that MDVs are stimulated upon various forms of mitochondrial stress, and the vesicles incorporated quantitative amounts of cargo, whose identity depended upon the nature of the stress. Under the conditions examined, MDVs did not incorporate complexes I and V, nor were any nucleoids present, demonstrating the specificity of cargo incorporation. Stress-induced MDVs are selectively enriched for oxidized proteins, suggesting that conformational changes induced by oxidation may initiate their incorporation into the vesicles. Ultrastructural analyses of MDVs isolated on sucrose flotation gradients revealed the formation of both single and double membranes vesicles of unique densities and uniform diameter. This work provides a framework for a reductionist approach towards a detailed examination of the mechanisms of MDV formation and cargo incorporation, and supports the emerging concept that MDVs are critical contributors to mitochondrial quality control.
We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature.
Methods and results
Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors.
Conclusions
The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases.
Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly. Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly. NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3) is the largest and the least understood of all the respiratory chain complexes. Mammalian complex I is composed of at least 46 subunits, which are encoded by both nuclear (39 subunits) and mitochondrial DNA (7 subunits) (1Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (686) Google Scholar, 2Skehel J.M. Fearnley I.M. Walker J.E. 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Mills D. Weiss H. Leonard K.R. J. Mol. Biol. 1997; 265: 409-418Crossref PubMed Scopus (133) Google Scholar) complex I show that the complex has an L-shaped form with one arm in the membrane and a peripheral arm protruding into the mitochondrial matrix. A second, horseshoe-shaped conformation of the E. coli complex I has also recently been proposed (8Bottcher B. Scheide D. Hesterberg M. Nagel-Steger L. Friedrich T. J. Biol. Chem. 2002; 277: 17970-17977Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The complex can be dissociated by treatment with detergent into three subcomplexes: Iα, corresponding to the peripheral arm and composed of ∼21 mostly hydrophilic subunits, and Iβ and Iγ, both of which make up the membrane arm (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar). Subcomplex Iα contains the NADH binding site and most of the redox centers. All of the mtDNA-encoded 1The abbreviations used are: mtDNA, mitochondrial DNA; BN-PAGE, Blue-Native PAGE; COX, cytochrome c oxidase; Alu, restriction endonuclease; Alu-FISH, Alu-PCR repeats fluorescence in situ hybridization; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. subunits and at least 16 of the nuclear-encoded subunits are found in the Iβ and Iγ fractions of the membrane arm. Deficiencies in complex I are among the most common respiratory chain defects (10Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2655) Google Scholar, 11Loeffen J. Elpeleg O. Smeitink J. Smeets R. Stockler-Ipsiroglu S. Mandel H. Sengers R. Trijbels F. van den Heuvel L. Ann. Neurol. 2001; 49: 195-201Crossref PubMed Scopus (165) Google Scholar, 12van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 13Triepels R. Smeitink J. Loeffen J. Smeets R. Trijbels F. van den Heuvel L. Hum. Genet. 2000; 106: 385-391Crossref PubMed Scopus (15) Google Scholar, 14Loeffen J. Smeitink J. Triepels R. Smeets R. Schuelke M. Sengers R. Trijbels F. Hamel B. Mullaart R. van den Heuvel L. Am. J. Hum. Genet. 1998; 63: 1598-1608Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 15Schuelke M. Smeitink J. Mariman E. Loeffen J. Plecko B. Trijbels F. Stockler-Ipsiroglu S. van den Heuvel L. Nat. Genet. 1999; 21: 260-261Crossref PubMed Scopus (243) Google Scholar). Mutations in the mtDNA-encoded subunits of complex I were the first to be associated with a respiratory chain disorder, Lebers hereditary optic neuropathy (10Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2655) Google Scholar, 16Wallace D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8739-8746Crossref PubMed Scopus (446) Google Scholar). Lebers hereditary optic neuropathy presents with the comparatively mild phenotype of adult-onset blindness due to optic nerve degeneration, and most cases are caused by mutations in one of four complex I subunit genes (MTND1, MTND4, MTND5, and MTND6) (17Wallace D.C. Singh G. Lott M.T. Hodge J.A. Schurr T.G. Lezza A.M. Elsas L.J. Nikoskelainen E.K. Science. 1988; 242: 1427-1430Crossref PubMed Scopus (2066) Google Scholar, 18Man P.Y. Turnbull D.M. Chinnery P.F. J. Med. Genet. 2002; 39: 162-169Crossref PubMed Scopus (387) Google Scholar). In contrast, the majority of early onset complex I deficiencies are severe, and often fatal, autosomal recessive disorders. DNA sequence analysis in more than 20 complex I patients has revealed mutations in seven structural genes: NDUFS2 (11Loeffen J. Elpeleg O. Smeitink J. Smeets R. Stockler-Ipsiroglu S. Mandel H. Sengers R. Trijbels F. van den Heuvel L. Ann. Neurol. 2001; 49: 195-201Crossref PubMed Scopus (165) Google Scholar); NDUFS4 (12van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 19Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (121) Google Scholar, 20Budde S.M. van den Heuvel L.P. Janssen A.J. Smeets R.J. Buskens C.A. DeMeirleir L. Van Coster R. Baethmann M. Voit T. Trijbels J.M. Smeitink J.A. Biochem. Biophys. Res. Commun. 2000; 275: 63-68Crossref PubMed Scopus (169) Google Scholar); NDUFS7 (13Triepels R. Smeitink J. Loeffen J. Smeets R. Trijbels F. van den Heuvel L. Hum. Genet. 2000; 106: 385-391Crossref PubMed Scopus (15) Google Scholar); NDUFS8 (14Loeffen J. Smeitink J. Triepels R. Smeets R. Schuelke M. Sengers R. Trijbels F. Hamel B. Mullaart R. van den Heuvel L. Am. J. Hum. Genet. 1998; 63: 1598-1608Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar); NDUFV1 (15Schuelke M. Smeitink J. Mariman E. Loeffen J. Plecko B. Trijbels F. Stockler-Ipsiroglu S. van den Heuvel L. Nat. Genet. 1999; 21: 260-261Crossref PubMed Scopus (243) Google Scholar, 21Benit P. Chretien D. Kadhom N. de Lonlay-Debeney P. Cormier-Daire V. Cabral A. Peudenier S. Rustin P. Munnich A. Rotig A. Am. J. Hum. Genet. 2001; 68: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); NDUFS1 (21Benit P. Chretien D. Kadhom N. de Lonlay-Debeney P. Cormier-Daire V. Cabral A. Peudenier S. Rustin P. Munnich A. Rotig A. Am. J. Hum. Genet. 2001; 68: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); and NDUFV2 (22Benit P. Beugnot R. Chretien D. Giurgea I. De Lonlay-Debeney P. Issartel J.P. Corral-Debrinski M. Kerscher S. Rustin P. Rotig A. Munnich A. Hum. Mutat. 2003; 21: 582-586Crossref PubMed Scopus (144) Google Scholar). Structural gene mutations were not found in about 50% of patients in these studies, suggesting that genes coding for assembly factors may be important causes of complex I deficiency. An assembly defect in complex I was described in a patient with a mutation in NDUFS4 (19Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (121) Google Scholar) and in a patient with a mutation in the mitochondrially encoded subunit ND4 (23Hofhaus G. Attardi G. EMBO J. 1993; 12: 3043-3048Crossref PubMed Scopus (88) Google Scholar). Saccharomyces cerevisiae is widely used as a model organism for mitochondrial respiratory chain function but does not contain a complex I (24Buschges R. Bahrenberg G. Zimmermann M. Wolf K. Yeast. 1994; 10: 475-479Crossref PubMed Scopus (28) Google Scholar, 25Nosek J. Fukuhara H. J. Bacteriol. 1994; 176: 5622-5630Crossref PubMed Google Scholar). The current model of assembly of complex I is based on pulse-chase labeling of assembly intermediates in N. crassa and on the characterization of assembly subcomplexes in N. crassa mutants (26Nehls U. Friedrich T. Schmiede A. Ohnishi T. Weiss H. J. Mol. Biol. 1992; 227: 1032-1042Crossref PubMed Scopus (76) Google Scholar, 27Tuschen G. Sackmann U. Nehls U. Haiker H. Buse G. Weiss H. J. Mol. Biol. 1990; 213: 845-857Crossref PubMed Scopus (107) Google Scholar, 28Kuffner R. Rohr A. Schmiede A. Krull C. Schulte U. J. Mol. Biol. 1998; 283: 409-417Crossref PubMed Scopus (89) Google Scholar, 29Ferreirinha F. Duarte M. Melo A.M. Videira A. Biochem. J. 1999; 342: 551-554Crossref PubMed Google Scholar, 30Duarte M. Sousa R. Videira A. Genetics. 1995; 139: 1211-1221Crossref PubMed Google Scholar); however, our knowledge of mammalian complex I assembly is very limited. Here, we report the identification of complex I subcomplexes in patients with complex I deficiency due to mutations in either nuclear or mitochondrial DNA and in a human/mouse hybrid cell line with a severe complex I deficiency due to an almost complete failure to assemble the holoenzyme complex. Using Blue-Native PAGE and a panel of antibodies specific for structural subunits of complex I, we identified a common set of complex I subcomplexes, and we propose a possible pathway for mammalian complex I assembly which differs significantly from that proposed for Neurospora. Characterization of Patients—At the time of biopsy, patient S was a 28-year-old male (described previously in Ref. 31Schwartz M. Vissing J. N. Engl. J. Med. 2002; 347: 576-580Crossref PubMed Scopus (488) Google Scholar) who suffered from lifelong exercise intolerance. Cardiac and pulmonary functions were normal, and he was otherwise well. Both parents and a 23-year-old sister were healthy. The isolated myopathy was associated with severe lactic acidosis, and a muscle biopsy revealed the presence of ragged-red fibers. Biochemical analysis of the patient's muscle revealed a selective complex I deficiency (<10% of control), whereas complexes II–IV had normal activity. The analysis of mtDNA showed a heteroplasmic 2-bp deletion in the MTND2 gene (90% of mtDNA in the patient muscle carries the mutation) causing a premature stop codon, which predicts a truncated protein. At the time of biopsy, patient G was a 20-year-old male with a lifelong history of severe aerobic exercise intolerance associated with exertional dyspnea and tachycardia. Biochemical studies of patient muscle biopsy revealed a selective decrease in complex I activity (<20% of control), whereas other respiratory chain activities were normal. Histochemical analysis revealed normal mitochondrial staining, with no ragged-red fibers. The patient's sister (23 years old) also suffers from a pure myopathy and severe exercise intolerance. Myoblasts from both siblings showed slightly decreased complex I activity (75% of control); fibroblasts from both siblings showed no abnormality. Fusion of myoblasts from both siblings with 143B.TK– rho0 cells rescued the complex I deficiency, indicating that the defect in patient G and his sister is of nuclear origin. Patients B1 and B2 are brothers, 39 and 38 years old, respectively, at the time of biopsy. They both suffer from mitochondrial encephalomyopathy and pigmentary retinopathy. Patient B1 had developmental problems, clumsiness, and borderline mental retardation since birth. He developed seizures at 19 years. Patient B2 has a lifelong history of learning disabilities. He had adult onset epilepsy and has a Wolff-Parkinson-White conduction defect. Muscle histochemistry showed ragged-red fibers in both patients, with increased SDH staining and no COX negative fibers. Complex I activity in muscle mitochondria from patient B1 was 11% and from patient B2 was 10% of the normal mean. All other respiratory chain enzyme activities were normal. Southern blot analysis of mtDNA from patient B1 muscle showed no evidence of a deletion, and sequencing of all the tRNA genes and all the mtDNA-encoded complex I genes showed no mutation. Low complex I activity was also found in fibroblasts from patient B1, and all other respiratory chain enzyme activities were normal. Fusion of patients fibroblasts with 143B.TK– rho0 cells rescued the complex I deficiency, indicating that the defect in patients B1 and B2 is of nuclear origin. Fusion of fibroblasts from patient B1 with fibroblasts from patient B2 showed low complex I in-gel activity, proving that they belong to the same genetic complementation group, as expected. Informed consent was obtained from all patients, and protocols were approved by the relevant Institutional Review Boards Cell Lines—Primary skin fibroblasts were established from biopsy material and immortalized by transduction with retroviral vectors expressing the type 16 human papilloma virus E7 gene and the catalytic component of human telomerase (32Morales C.P. Holt S.E. Ouellette M. Kaur K.J. Yan Y. Wilson K.S. White M.A. Wright W.E. Shay J.W. Nat. Genet. 1999; 21: 115-118Crossref PubMed Scopus (689) Google Scholar). The fibroblasts were grown in high glucose Dulbecco's modified Eagle's medium containing 110 mg/ml pyruvate and 10% fetal bovine serum. The LM.TK– rho0 mouse cell line (a kind gift of Dr. Eric Schon) was grown in high glucose Dulbecco's modified Eagle's medium containing 110 mg/ml pyruvate, 10% fetal bovine serum, and 50 μg/ml uridine. Cell Fusion—LM.TK– rho0 cells were infected with a retroviral construct conferring resistance to puromycin (33Miller A.D. Miller D.G. Garcia J.V. Lynch C.M. Methods Enzymol. 1993; 217: 581-599Crossref PubMed Scopus (378) Google Scholar). Control fibroblasts were plated together with LM.TK– rho0 cells and fused 16 h later using polyethylene glycol-1500:Me2SO (40%:10%). The fused cells were selected in medium containing 1 μg/ml puromycin and 400 μg/ml G-418 for 3 weeks. Muscle Tissue, Preparation of Mitochondria—Muscle biopsies from patients S, G, B1, and B2 and controls were obtained and stored frozen at –80 °C. Muscle homogenates (5%) prepared in 150 mm KCl, 20 mm Tris-HCl, 2 mm EDTA (pH 7.5) were centrifuged twice for 10 min at 600 × g to obtain postnuclear supernatant. Mitochondria were pelleted by centrifugation for 20 min at 10,000 × g. Protein concentration was measured by the Bradford method (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222903) Google Scholar). Electrophoretic Methods—Mitoplasts were prepared by treatment with 0.8 mg (fibroblasts) or 1.2 mg (myoblasts) of digitonin/mg of protein, as described previously (35Klement P. Nijtmans L.G. Van den Bogert C. Houstek J. Anal. Biochem. 1995; 231: 218-224Crossref PubMed Scopus (94) Google Scholar). Mitoplasts or mitochondria were solubilized with 1% lauryl maltoside, and 10–20 μg of solubilized protein was used for electrophoresis. BN-PAGE (36Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1937) Google Scholar) was used for separation of samples in the first dimension on 6–15% or 5–12% polyacrylamide gradients. In-gel qualitative assays for complex I and cytochrome c oxidase (COX) activity were performed as described (37Zerbetto E. Vergani L. Dabbeni-Sala F. Electrophoresis. 1997; 18: 2059-2064Crossref PubMed Scopus (255) Google Scholar). For the two-dimensional analysis, strips of the first dimension gel were incubated for 45 min in 1% SDS and 1% β-mercaptoethanol, and then 10% polyacrylamide SDS-PAGE was used to separate the proteins in the second dimension (38Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10754) Google Scholar). Respiratory complexes I–IV were detected by Western blot analysis using monoclonal and polyclonal antibodies. Anti-COX IV, anti-complex II/70 kDa, anti-complex III/Core1, and complex I antibodies against subunits 30, 20, and 14 (Table I) were from Molecular Probes, Eugene, OR. Other antibodies against complex I subunits were kindly provided by Dr. R. Capaldi (39, 15, and 8 kDa); Dr. B. Robinson (49 and 17 kDa), Dr. J. Walker (24 kDa), Dr. A. Lombes (ND1), and Dr. V. Petruzzella (18 kDa).Table IComplex I subunits described in this studyNameaThe names of the nuclear-encoded subunits used in this study are based on the apparent molecular size.Gene nameLocalization49 kDaNDUFS2Peripheral arm, Iα subcomplex39 kDaNDUFA9Peripheral and membrane arm boundary, Iα and Iγ subcomplexes30 kDaNDUFS3Peripheral arm, Iα subcomplex24 kDaNDUFV2Peripheral arm, Iα subcomplex20 kDaNDUFS7Peripheral arm, Iα subcomplex18 kDaNDUFS4Peripheral arm, Iα subcomplex17 kDaNDUFB6Membrane arm, Iβ subcomplex15 kDaNDUFS5Peripheral and membrane arm boundary, Iα and Iγ subcomplexes14 kDaNDUFA6Peripheral arm, Iα subcomplex8 kDaNDUFA2Peripheral arm, Iα subcomplexND1MTND1Membrane arm, Iγ subcomplexa The names of the nuclear-encoded subunits used in this study are based on the apparent molecular size. Open table in a new tab Identification of Complex I Subcomplexes—BN-PAGE analysis of mitochondria from muscle biopsies of patients S, G, B1, and B2 showed decreased in-gel activity of complex I, whereas the activity of COX was normal (Fig. 1A). Western blot analysis showed normal levels of immunodetectable COX, complex II (not shown), and complex III; however, fully assembled complex I was markedly decreased in all four patients (Fig. 1B). Interestingly, antibodies against both nuclear (39 and 49 kDa) and mitochondrial (ND1) subunits of complex I identified several common subcomplexes with apparent molecular masses of 250, 310, 380, 480, and 650 kDa (Fig. 1B, Table II). An additional subcomplex of about 830 kDa was seen only in patients B1 and B2. Although the pattern of the subcomplexes was similar in all four patients, the intensities of immunodetectable signals varied among the patients. The ratio between the unassembled (found in subcomplexes) and the fully assembled subunits in patients B1 and B2 was lower than in patients S and G, in whom the amount of ND1 and 49-kDa subunits in the subcomplexes was similar or higher than in the fully assembled complex.Table IISubunit composition of complex I subcomplexesSubcomplexMSaMS, molecular size.Subunit4939ND115302014182417bAntibody-detected pattern of the 17-kDa subunit presented as a streak and could not be assigned to any subcomplex.8kDaI200XXXII230XXXIII250XXXIV310XXXXV380XXXXXVI480XXXXXVII650XXXXXXXXXXFully assembled950XXXXXXXXXXXa MS, molecular size.b Antibody-detected pattern of the 17-kDa subunit presented as a streak and could not be assigned to any subcomplex. Open table in a new tab Subunit Composition of Complex I Subcomplexes—To determine the subunit composition of the complex I subcomplexes, we used a set of 11 antibodies directed against structural subunits of complex I (described under "Experimental Procedures") in combination with two-dimensional electrophoretic analysis. Muscle mitochondria (20 μg of protein) from patient S were analyzed using BN-PAGE in the first dimension and Tricine/SDS-PAGE in the second dimension. Fig. 2 shows markedly decreased amounts of individual subunits in the fully assembled complex I from the patient when compared with control. Seven distinguishable subcomplexes were present in patient S mitochondria (marked I–VII). In addition to the five subcomplexes described in the one-dimensional gels above, subcomplexes of molecular mass 230 and 200 kDa were identified (Table II). A largely similar pattern was seen in muscle mitochondria from patients G, B1 (Fig. 3), and B2 (data not shown), although the relative intensities varied. The specific 830-kDa subcomplex of patient B1 mitochondria contained all analyzed subunits (49, 39, 15, 30, 20, 14, and 18 kDa and ND1). In all patients, antibodies against subunits 49 and 30 kDa detected these subunits in all the subcomplexes except for subcomplex II. A similar pattern was found for subunit 39 kDa; however, it appeared more as a streak than strong individual subcomplexes. A second pattern was seen using antibodies to subunits 24, 20, and 18 kDa, which were present in subcomplexes II and VII. The 8-kDa subunit and ND1 also shared a similar pattern and were not present in the lower molecular mass subcomplexes. Although the 24-kDa antibody does detect some subcomplexes in the control, the ratio between the fully assembled enzyme and the subcomplexes is substantially higher than seen in the patient. The 14- and 15-kDa subunits were only detected in the 650-kDa subcomplex (VII) (and the 830-kDa subcomplex unique to patients B1 and B2), and the 17-kDa subunit formed a very weak streak that could not be linked to any of the subcomplexes. Subcomplexes of complex I were also detected in fibroblasts from patient B1 (data not shown); however, the intensities were markedly decreased in comparison with those seen in muscle mitochondria. In control mitochondria, a weak 650-kDa subcomplex could be detected with antibodies against the ND1 and 18-kDa subunits (Fig. 2); however, the other subcomplexes seen in the patients were not observed even after long exposure of the control immunoblot.Fig. 3Identification and subunit composition of complex I subcomplexes in patients G and B1. Muscle mitochondria from patient G and patient B1 (20 μg of protein) were analyzed by two-dimensional PAGE, and the presence of individual complex I subunits in the subcomplexes was determined by immunoblotting. The filled line marks the position of fully assembled complex I, and the dashed lines indicate subcomplexes I–VII. The patient B1 specific subcomplex of 830 kDa is marked with a filled line. The migration of molecular mass standards (in kDa) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Presence of Complex I Subcomplexes in a Human/Mouse Hybrid Cell Line—A standard method to determine whether a respiratory chain defect maps to nuclear or mitochondrial DNA is to fuse patient cells with rho0 cells to test for complementation. Complementation of the biochemical phenotype in this experiment indicates a recessive nuclear gene defect. To test whether mouse nuclear genes could rescue human nuclear respiratory chain gene deficiencies, we previously carried out this experiment in several patient cell lines, but never observed complementation. In fact, fusion of a control human fibroblast line with mouse LM.TK– rho0 cells resulted in severely decreased COX and complex I activities. BN-PAGE analysis showed a strong dominant-negative effect of the mouse nucleus on complex I assembly, whereas complex II was normal (Fig. 4A). This phenotype does not arise from a loss of human chromosomes as Alu-FISH analysis shows a normal diploid chromosome number on metaphase spreads (data not shown). Rather, it appears to result from the dominant negative effects of specific mouse genes present in the human nuclear and mitochondrial background. Two-dimensional analysis of the fusion cells showed the presence of complex I subcomplexes (Fig. 4B) with apparent molecular masses of 650, 480, 380, 310, and 250 kDa, corresponding to subcomplexes III–VII. Almost no fully assembled complex could be detected in these cells, and the subunit pattern in the complex I subcomplexes was similar to that found in complex I patients. Blue-Native PAGE is a powerful technique for the analysis of respiratory chain complexes and for detecting and diagnosing respiratory chain disorders (39Nijtmans L.G. Henderson N.S. Holt I.J. Methods (Orlando). 2002; 26: 327-334Google Scholar). Pulse-chase labeling of mitochondrial proteins with [35S]methionine, together with BN-PAGE, allowed the identification of three intermediates in the assembly of COX in mammalian cells (40Nijtmans L.G. Taanman J.W. Muijsers A.O. Speijer D. Van den Bogert C. Eur. J. Biochem. 1998; 254: 389-394Crossref PubMed Scopus (205) Google Scholar). Patients with Leigh syndrome caused by mutations in SURF1 show a characteristic early assembly defect (41Tiranti V. Jaksch M. Hofmann S. Galimberti C. Hoertnagel K. Lulli L. Freisinger P. Bindoff L. Gerbitz K.D. Comi G.P. Uziel G. Zeviani M. Meitinger T. Ann. Neurol. 1999; 46: 161-166Crossref PubMed Scopus (113) Google Scholar) in which COX assembly is arrested before the incorporation of subunit II into the catalytic core, resulting in the accumulation of two early assembly intermediates whose presence discriminates between COX-deficient SURF1 and non-SURF1 patients (42Coenen M.J. van den Heuvel L.P. Nijtmans L.G. Morava E. Marquardt I. Girschick H.J. Trijbels F.J. Grivell L.A. Smeitink J.A. Biochem. Biophys. Res. Commun. 1999; 265: 339-344Crossref PubMed Scopus (49) Google Scholar). BN-PAGE has also been used to characterize the assembly of Tom7, a component of the preprotein translocase complex in the outer mitochondrial membrane (43Johnston A.J. Hoogenraad J. Dougan D.A. Truscott K.N. Yano M. Mori M. Hoogenraad N.J. Ryan M.T. J. Biol. Chem. 2002; 277: 42197-42204Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Here, we have used BN-PAGE and a panel of structural subunit antibodies to identify complex I subcomplexes in patients with either nuclear or mtDNA complex I defects. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes, albeit with very different relative abundances, suggests that the subcomplexes represent stalled intermediates in the assembly of the holoenzyme complex. Antibodies against the 39-kDa, 49-kDa, and ND1 subunits of complex I identified five distinct subcomplexes (250, 310, 380, 480, and 650 kDa) in the one-dimensional BN-PAGE in muscle mitochondria from the four patients studied, and two-dimensional PAGE revealed two additional lower molecular mass subcomplexes (200 and 230 kDa). As the respiratory chain complexes maintain their native conformation on the one-dimensional PAGE, some of the subunits will not be accessible to the antibodies, and the inability to detect these two smaller subcomplexes on the one-dimensional PAGE likely reflects this limitation. Although the relative intensities of the subcomplexes varied considerably among the patients, a consistent pattern was observed. In general, complex I subunits could be divided into four groups. Subunits 49, 39, and 30 kDa, all part of the Iα structural subcomplex described by Sazanov et al. (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar), were present in all subcomplexes, except subcomplex II. The 39-kDa subunit is also found in the Iγ subcomplex, indicating that it is probably found on the periphery of the complex at the boundary between the peripheral and the membrane arms (Table I). The streaky nature of the antibody detection pattern of the 39-kDa subunit in the second dimension gel suggests a weak attachment to the other subunits in the subcomplexes. Subunits 24, 20, and 18 kDa, which form part of the peripheral arm Iα subcomplex, were present in subcomplexes II and VII. Subcomplex II, with a molecular mass of ∼230 kDa, did not contain any of the other subunits studied here. Subunits ND1 and 8 kDa showed a similar distribution among the subcomplexes, although they belong to different parts of the holoenzyme complex, the membrane and peripheral arms, respectively. They were both detected in subcomplexes V–VII, and the ND1 subunit was also found in subcomplex IV. The last group contains the 15- and 14-kDa subunits, which are part of subcomplex VII. The 17-kDa subunit, described as belonging to the membrane Iβ subcomplex (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar), appeared as a very faint streak on the immunoblot and was the only subunit that we were not able to assign to any subcomplex. The additional subcomplex of about 830 kDa observed in mitochondria from patients B1 and B2 was not found in fibroblasts or muscle from 10 other complex I patients (data not shown). Two-dimensional PAGE showed the presence of ND1, 49-, 39-, 30-, 20-, 18-, 15-, and 14-kDa subunits in the 830-kDa subcomplex with intensities similar to those found in the fully assembled complex, suggesting a late assembly defect in patients B1 and B2. Triepels et al. (44Triepels R.H. Hanson B.J. van den Heuvel L.P. Sundell L. Marusich M.F. Smeitink J.A. Capaldi R.A. J. Biol. Chem. 2001; 276: 8892-8897Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) studied complex I subcomplexes in several patients with complex I deficiency using a standard immunoblot analysis to measure the steady-state levels of individual structural subunits (which would include free subunits and those in partially or fully assembled complexes) and sucrose gradient centrifugation to investigate assembly patterns. They divided the subunits into three classes depending on their expression levels and complex I activity in the studied patients: expression levels of 39- and 30-kDa subunits most closely paralleled the loss of activity; levels of 20- and 18-kDa subunits were higher and levels of 15- and 8-kDa subunits were lower than predicted from the activity measurement. In this study, we also assigned 39- and 30-kDa subunits and 20- and 18-kDa subunits into two separate groups based on their presence in complex I subcomplexes; however, the 15- and 8-kDa subunits belong to different groups in our study. Several studies have focused on determining the role of mtDNA-encoded subunits of complex I on enzyme assembly. The deletion of human ND4 subunit caused a failure of other mtDNA-encoded subunits to assemble; however, the nuclear subunits involved in the redox reactions appeared to form an intermediate with normal NADH:Fe(CN)6 oxidoreductase activity (23Hofhaus G. Attardi G. EMBO J. 1993; 12: 3043-3048Crossref PubMed Scopus (88) Google Scholar). A similar result has been reported in an ND4 mutant in plants, where a subcomplex with NADH dehydrogenase activity was characterized (45Karpova O.V. Newton K.J. Plant J. 1999; 17: 511-521Crossref Scopus (61) Google Scholar). MtDNA subunits ND2 and ND3 are required for the membrane arm assembly in N. crassa (46Alves P.C. Videira A. Biochem. Cell Biol. 1998; 76: 139-143Crossref PubMed Scopus (14) Google Scholar), and the ND6 protein is necessary for the membrane arm assembly in mouse mitochondria (47Bai Y. Attardi G. EMBO J. 1998; 17: 4848-4858Crossref PubMed Scopus (172) Google Scholar). In contrast, a lack of the ND5 subunit does not influence complex I assembly in humans (48Hofhaus G. Attardi G. Mol. Cell. Biol. 1995; 15: 964-974Crossref PubMed Google Scholar). In the unicellular green alga Chlamydomonas reinhardtii, the absence of the ND1 or ND6 subunit prevented the assembly of complex I, whereas the loss of the ND4 or ND4/ND5 led to the formation of a 650-kDa subcomplex with NADH dehydrogenase activity (49Cardol P. Matagne R.F. Remacle C. J. Mol. Biol. 2002; 319: 1211-1221Crossref PubMed Scopus (107) Google Scholar). The loss of the ND2 protein in patient S compromised the assembly of complex I and led to the formation of complex I subcomplexes; however, NADH dehydrogenase activity could not be detected in these subcomplexes by an in-gel activity stain. This indicates a significant role for the ND2 subunit in the assembly and/or stability of complex I. The location of the ND2 subunit in the center of Iγ subcomplex in the vicinity of the ND1 protein (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar) suggests that the absence of this subunit causes the disruption of the complex in patient mitochondria. Much less is known about the role of nuclear-encoded subunits of mammalian complex I in the assembly of the complex. A patient with a mutation in the NDUFS4 gene encoding the 18-kDa subunit was unable to assemble complex I (19Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (121) Google Scholar), but no subcomplexes were described in that case. Interestingly, when nuo21, the NDUFS4 orthologue in N. crassa, was disrupted, the mutant was able to assemble an almost intact enzyme. Subcomplexes of ∼500 and 200 kDa, containing the 20- and 39-kDa subunits, respectively (44Triepels R.H. Hanson B.J. van den Heuvel L.P. Sundell L. Marusich M.F. Smeitink J.A. Capaldi R.A. J. Biol. Chem. 2001; 276: 8892-8897Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), were described in a patient suggested to have a mutation in a complex I assembly factor. The current model of complex I biogenesis and assembly is based on studies in N. crassa, which contains about 35 subunits, at least three of which are not found in mammalian complex I and whose function remains unknown (50Videira A. Duarte M. Biochim. Biophys. Acta. 2002; 1555: 187-191Crossref PubMed Scopus (81) Google Scholar, 51Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. J. Biol. Chem. 2002; 277: 50311-50317Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The identification and characterization of complex I subcomplexes in this study allow us to propose a model for complex I assembly in humans (Fig. 5) that differs significantly from that proposed for N. crassa. Four assembly intermediates have been identified in the N. crassa enzyme: the peripheral arm, a large and a small subcomplex of the membrane arm, and the entire membrane arm itself (52Schulte U. J. Bioenerg. Biomembr. 2001; 33: 205-212Crossref PubMed Scopus (55) Google Scholar). The mature complex is thought to assemble by stepwise association of these modules. Disruption of specific complex I structural subunits disturbs holoenzyme assembly, resulting in the accumulation of one or more of these enzyme subcomplexes (53Videira A. Duarte M. J. Bioenerg. Biomembr. 2001; 33: 197-203Crossref PubMed Scopus (34) Google Scholar); however, mutations in subunits belonging to one arm do not interfere with assembly of the other arm. On the contrary, they lead to the accumulation of the other subcomplexes of the enzyme. Subcomplexes of the peripheral arm have not been observed, nor have subcomplexes involving the peripheral arm and part of the membrane arm. The results of our study on the human enzyme stand in sharp contrast to this picture: many more assembly intermediates are observed, including subcomplexes of the peripheral arm and subcomplexes containing parts of both arms, suggesting that the peripheral and membrane arms are not assembled in separate, independent pathways. In our model, the peripheral arm of mammalian complex I is assembled as two independent units (subcomplexes I and II). Subcomplex I associates with other subunits, forming subcomplex III, which is in turn attached to subunit(s) that are part of the membrane arm, forming subcomplex IV. Since ND1 subunit is a hydrophobic integral membrane protein, we can speculate that this subunit is inserted into the membrane prior to its association with the subcomplex III, hence subcomplex IV, which contains part of the peripheral arm and part of the membrane arm, is membrane-bound. Additional subunits are added to this subcomplex, resulting in subcomplexes V and VI. The next step of assembly requires coupling of subcomplex II (which forms part of the peripheral arm) and subcomplex VI and addition of other subunits, resulting in the formation of subcomplex VII. The remaining subunits are further associated with subcomplex VII and produce a fully assembled complex. Two chaperones, CIA30 and CIA84, play an essential role in the assembly of the membrane arm of the N. crassa complex I, interacting with the large subcomplex of the membrane arm and promoting its association with the smaller component to form the entire membrane arm (28Kuffner R. Rohr A. Schmiede A. Krull C. Schulte U. J. Mol. Biol. 1998; 283: 409-417Crossref PubMed Scopus (89) Google Scholar). A ubiquitously expressed human homologue of only one of these, CIA30, has been described (54Janssen R. Smeitink J. Smeets R. van Den Heuvel L. Hum. Genet. 2002; 110: 264-270Crossref PubMed Scopus (58) Google Scholar), but its function is not yet known, and DNA sequence analysis of 13 candidate complex I patients (with putative assembly defects) has not revealed any mutations in this gene (54Janssen R. Smeitink J. Smeets R. van Den Heuvel L. Hum. Genet. 2002; 110: 264-270Crossref PubMed Scopus (58) Google Scholar). We acknowledge the contributions of Drs. N. Buist, R. Weleber, M. Salinsky, R. Steiner, and C. Williams to the care of patients B1 and B2. We thank Dr. M. Chevrette for the Alu-FISH analysis and T. Johns for excellent technical assistance.
Mitochondria interact with the ER at structurally and functionally specialized membrane contact sites known as mitochondria–ER contact sites (MERCs). Combining proximity labelling (BioID), co-immunoprecipitation, confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. BioID analyses using ER-targeted, outer mitochondrial membrane-targeted, and MERC-targeted baits, confirmed the presence of this complex at MERCs and the specificity of the interaction. Deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs. Loss of the ESYT1–SYNJ2BP complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by reexpression of WT ESYT1 and an artificial mitochondria–ER tether. Together, these results reveal a novel function for ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.
Rats were fed a diet containing 1% beta-guanidino-propionic acid (GPA) for 6-12 wk to deplete their muscles of phosphocreatine (PCr). Gated 31P nuclear magnetic resonance (NMR) spectra were obtained from the gastrocnemius-plantaris muscle at various time points during either a 1- or 3-s isometric tetanic contraction using a surface coil. The energy cost of a 1-s tetanus in unfatigued control rat muscle was 48.4 mumol ATP X g dry wt-1 X s-1 and was largely supplied by PCr; anaerobic glycogenolysis was negligible. In GPA-fed rats PCr was undetectable after 400 ms. This had no effect on initial force generated per gram, which was not significantly different from controls. Developed tension in a 3-s tetanus in GPA-fed rats could be divided into a peak phase (duration 0.8-0.9 s) and a plateau phase (65% peak tension) in which PCr was undetectable and the [ATP] was less than 20% of that in control muscle. Energy from glycogenolysis was sufficient to maintain force generation at this submaximal level. Mean net glycogen utilization per 3-s tetanus was 78% greater than in control muscle. However, the observed decrease in intracellular pH was less than that expected from energy budget calculations, suggesting either increased buffering capacity or modulation of ATP hydrolysis in the muscles of GPA-fed rats. Our results demonstrate that the transport role of PCr is not essential in contracting muscle in GPA-fed rats. PCr is probably important in this regard in the larger fibers of control muscle. Although fast-twitch muscles depleted of PCr have nearly twice the glycogen reserves of control muscle, glycogenolysis is limited in its capacity to fill the role of PCr as an energy buffer under conditions of maximum ATP turnover.
Mitochondrial (mt)DNA is strictly maternally inherited in mammals; new mutations thus segregate along maternal lineages without the benefit of homologous recombination with mtDNA of paternal origin. Despite the high mtDNA copy number (∼100 000 or more) in mature oocytes, and despite the relatively small number of cell divisions during oogenesis, mtDNA sequence variants segregate rapidly between generations. This paradoxical behaviour has been ascribed to the presence of a mtDNA 'bottleneck' in oogenesis or early embryogenesis. The nature and size of this bottleneck have been the subject of much controversy. This review argues that segregation of mtDNA sequence variants in the female germline occurs primarily during mitosis in the oocyte precursor population. Segregation is rapid because the precursor cells (primordial germ cells and oogonia) contain a relatively small number of mtDNA templates (the bottleneck) and because the replication of mtDNA is under relaxed control. For the most part, the process appears similar in mice segregating polymorphic sequence variants and in human pedigrees segregating pathogenic point mutations. In particular, there is no evidence for selection against high levels of pathogenic mtDNA point mutations in oogenesis, in early embryonic development, or in fetal development, thus suggesting that efficient respiratory chain function is not critical until post-natal life. These results have important practical implications for clinical genetics.
Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form. Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form. Identifying and studying the processes and the genes that are involved in determining the rate of aging is a challenging area of modern genetics. In particular, it would be of interest to determine whether the activity of specific genes limits human life span. Several epidemiological studies of centenarians are being carried out with this goal in mind under the hypothesis that there might be a genetic basis for the exceptional life span of very long lived individuals (1Perls T. Terry D.F. Silver M. Shea M. Bowen J. Joyce E. Ridge S.B. Fretts R. Daly M. Brewster S. Puca A. Kunkel L. Results Probl. Cell Differ. 2000; 29: 1-20Crossref PubMed Scopus (10) Google Scholar). However, given the pervasive evolutionary conservation of physiological processes among organisms, a practical approach to find genes that might be involved in human aging is to first investigate the genetic basis of aging in lower organisms. The nematode genetic model system, Caenorhabditis elegans, is being extensively used to this end, and a number of genes that have been identified in this organism for their effect on aging are now also being studied in vertebrates (2Hekimi S. Burgess J. Bussiere F. Meng Y. Benard C. Trends Genet. 2001; 17: 712-718Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Tissenbaum H.A. Guarente L. Dev. Cell. 2002; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The clk-2 mutants of C. elegans display a pleiotropic phenotype (reviewed in Ref. 4Benard C. Hekimi S. Mech. Ageing Dev. 2002; 123: 869-880Crossref PubMed Scopus (5) Google Scholar) that includes a slowing down of numerous physiological processes, including embryonic and postembryonic development, behavioral rates, and reproduction (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). clk-2 mutants also show an increase in life span that is particularly dramatic in combination with mutations in other genes, such as clk-1 and daf-2 (2Hekimi S. Burgess J. Bussiere F. Meng Y. Benard C. Trends Genet. 2001; 17: 712-718Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 6Lakowski B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (425) Google Scholar). The clk-2 mutations are temperature-sensitive (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar, 7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and at 25 °C produce a lethal embryonic phenotype resulting in differentiated but highly disorganized embryos (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). This is likely to be the null phenotype, since it is also produced by RNA interference at all temperatures. Extensive temperature shift experiments have demonstrated that clk-2 is required for embryonic development only during a narrow time window in which oocyte maturation, fertilization, the completion of meiosis, and the initiation of embryonic development occurs (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). However, these events as well as subsequent embryonic development appear to proceed entirely normally until the 100-cell stage, after which aberrant development becomes apparent. Surprisingly, all clk-2 phenotypes, including the phenotypes observed in adults that are ∼1000 times larger than the eggs produced by the mother, are rescued by a maternal effect; i.e. homozygous mutant animals, issued from a heterozygous mother, appear wild type. This maternal rescue effect suggests that the presence of maternally provided clk-2 product might induce a self-maintained epigenetic state, although the possibility that the maternally provided clk-2 product can still function efficiently after extreme dilution cannot be excluded. A number of cellular phenotypes of clk-2 mutants have also been identified in addition to the organismal phenotypes described above (7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Gartner A. Milstein S. Ahmed S. Hodgkin J. Hengartner M.O. Mol. Cell. 2000; 5: 435-443Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). For example, the germ lines of clk-2 mutants do not respond normally to ionizing radiation. In the wild type, irradiation leads to cell cycle arrest in the mitotic phase of the germ line and to apoptotic cell death in the meiotic phase of the germ line. Both of these responses are abolished in clk-2 mutants. In addition, clk-2 mutants fail to respond with cell cycle arrest to treatment with hydroxyurea (HU), 1The abbreviations used are: HU, hydroxyurea; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GST, glutathione S-transferase. a drug that blocks DNA replication, suggesting a defect in the S-phase replication checkpoint. Taken together, these cellular phenotypes suggest that clk-2 mutants are defective in important aspects of the normal cellular response to DNA damage. The C. elegans clk-2 gene encodes a protein of 877 amino acids that is similar to Saccharomyces cerevisiae Tel2p and has a unique homologue in every eukaryotic genome sequenced to date (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). Yeast cells carrying the hypomorphic tel2-1 mutation grow slowly and have short telomeres (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). The telomeres shorten gradually in the tel2 cells, reaching their shortest lengths only after ∼150 generations. In addition to affecting the length of telomeres, Tel2p has also been shown to be involved in the telomere position effect, contributing to silencing of subtelomeric regions. Mutations in other genes, such as tel1, that also affect telomere length do not result in abnormal telomere position effect, indicating that the telomere position effect defect in tel2 mutants is not a simple consequence of the altered telomere length (10Zakian V.A. Annu. Rev. Genet. 1996; 30: 141-172Crossref PubMed Scopus (170) Google Scholar). In contrast to the viable tel2–1 mutants, cells that fully lack Tel2p die rapidly with an abnormal cell morphology, which suggests that Tel2p also has telomere-independent functions in yeast (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). Worm clk-2 mutants also have altered telomere length. In contrast to the phenotype in yeast, clk-2(qm37) mutants have lengthened telomeres, and transgenic expression of clk-2 shortens some telomeres (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). Although yeast Tel2p can bind single- and double-stranded DNA and RNA under some in vitro conditions (11Kota R.S. Runge K.W. Nucleic Acids Res. 1998; 26: 1528-1535Crossref PubMed Scopus (29) Google Scholar, 12Kota R.S. Runge K.W. Chromosoma. 1999; 108: 278-290Crossref PubMed Scopus (22) Google Scholar), a functional worm CLK-2::GFP fusion protein accumulates predominantly in the cytoplasm (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). These findings, together with the broad pleiotropy observed mostly in worms, but also in yeast, suggest that clk-2 and tel2 mutations affect telomere length indirectly. Cell Culture—All cells were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (plus nonessential medium amino acids for HT-1080 and SK-HEP-1) at 37 °C in an atmosphere of 5% CO2 and 95% air. Construction of the Plasmid pLXSH-hclk2 and Establishment of a Stable Cell Line Overexpressing hCLK2—A cDNA clone hk02952 (insert size 4337 bp), containing the full-length hclk2 cDNA sequence, as well as parts of intronic sequences (1929–2171, 2288–2456, and 2812–3434) was obtained from Kazusa DNA Research Institute, Japan. Using this cDNA as a template, two fragments that exclude the intron sequences, Δhclk2-A (from bp 256 to 1929) and Δhclk2-B (from bp 1929 to 3434) were generated by PCR and cloned into a pcDNA3.1/V5/His/TOPO vector (Invitrogen) to produce pcDNA3.1-Δhclk2-A and pcDNA3.1-Δhclk2-B. A BamHI-EcoRV fragment from pcDNA3.1-Δhclk2-A(–) was subcloned into the BamHI–HpaI site of pLXSH (13Miller A.D. Buttimore C. Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1144) Google Scholar) to produce pLXSH-Δhclk2-A. A BamHI fragment from pcDNA3.1-Δhclk2-A(–) was inserted into the BamHI site of pLXSH-Δhclk2-A to produce pLXSH-hclk2. Stable virus-producing cell lines were generated using procedures described previously (14Miller A.D. Miller D.G. Garcia J.V. Lynch C.M. Methods Enzymol. 1993; 217: 581-599Crossref PubMed Scopus (378) Google Scholar). Briefly, the retroviral constructs were used to transfect GP + E86 ecotropic packaging cells (15Markowitz D. Goff S. Bank A. J. Virol. 1988; 62: 1120-1124Crossref PubMed Google Scholar), and viruses thus produced were used to infect the amphotropic packaging cell line PA317. Selection was performed 48 h after infection in 400 units/ml hygromycin B and continued until colonies were visible. The colonies were pooled and expanded to establish the virus-producing cell lines. Target cells (see Table I) were transduced with the retrovirus as described (16Lochmuller H. Johns T. Shoubridge E.A. Exp. Cell Res. 1999; 248: 186-193Crossref PubMed Scopus (83) Google Scholar) and selected in hygromycin at the concentrations indicated. All surviving cells were kept together as a pool, which constitutes the SK-HEP-1-overexpressing hCLK2 cell line.Table ICell lines infectedName (ATCC No)Tissue derivationHygromycinunits/mlC2C12 (CRL-1772)Mouse myoblast400Rat1-R12 (CRL-2210)Rat fibroblast200A549 (CCL-18S)Human lung carcinoma900SK-N-ASHuman neuroblastoma400SK-HEP-1Human liver adenocarcinoma400HT-1080Human fibrosarcoma400293Human kidney carcinoma400MCH58Human fibroblast100 Open table in a new tab Construction of Plasmid pTRE2-hclk2 and Establishment of a Double Stable Tet-off HT-1080 Line with Inducible hCLK2—The hclk2 full-length cDNA sequence containing engineered NotI and EcoRV sites was generated by PCR and inserted into the NotI–EcoRV site of pTRE2 (Clontech, Palo Alto, CA) to produce plasmid pTRE2-hclk2. The plasmid DNA was transfected into premade Tet-off HT-1080 cells (Clontech) using the superfect reagent (Qiagen). Cells were selected in 400 units/ml hygromycin 48 h after infection, and selection was continued until colonies were visible. 30 colonies were picked, and immunoblot analysis using anti-hCLK2 antibodies (see below) showed that five clones (numbers 3, 6, 11, 19, and 21) overexpressed hCLK2 in an inducible manner. Cells were grown in the presence of doxycycline (1 μg/ml) to turn off hCLK2 expression and grown in the absence of doxycycline to turn on hCLK2 expression. Immunoblot analysis showed that the level of expression of hCLK2 in the Tet-off cell line (clone 21) was dependent on the dosage of doxycycline. Knocking Down the Expression of hCLK2 by Sequence-specific Small Interfering RNA (siRNA)—siRNA oligonucleotides were synthesized by Dharmacon Research (Lafayette, Colorado). siRNA duplex selection and transfection were performed as described (17Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8185) Google Scholar). The sequence of the siRNA for targeting endogenous hclk2 was as follows: sense siRNA, 5′-GCGGUAUCUCGGUGAGAUGdT-3′; antisense siRNA, 5′-CAUCUCACCGAGAUACCGCdT-3′. For each well of a six-well plate, 240 pmol of siRNA duplex was used. Cells were exposed to the siRNA treatment on day 1, and they were passaged 1:4 on day 4. Preparation of Antibodies Directed against hCLK-2 Protein—Two separate antigens were used to develop anti-hCLK2 polyclonal antibodies. The first antigen was generated as follows. A PCR fragment corresponding to bases 1516–1929 of the hclk2 clone hk02952, encoding amino acids 414–551 of hCLK2, was cloned into the pGEX-3X expression vector (Amersham Biosciences). A GST-hCLK2-(414–551) protein of the expected size (∼46 kDa) was expressed in DH10b bacteria and purified by affinity chromatography on a GST slurry. This recombinant protein was injected into two rabbits (2779 and 2780) to obtain polyclonal antibodies. To generate the second antigen, a PCR fragment corresponding to bases 279–1519 of the hclk2 clone hk02952, encoding amino acids 2–415 of hCLK2, was cloned into the pGEX-3X expression vector. A GST-hCLK2-(2–415) protein of the expected size (∼78 kDa) was expressed in DH10b bacteria and was purified from bacterial inclusion bodies. This recombinant protein was injected into two rabbits (2838 and 2839) to obtain polyclonal antibodies. All four sera specifically react to hCLK2 by the following criteria. The terminal bleed of each rabbit recognizes the corresponding bacterial antigen, in vitro translated hCLK2, a band at the expected size of ∼100 kDa in cell extracts, and a strong band of the same size in cells overexpressing hCLK2 (see Table I). This ∼100-kDa band is not detected by any of the preimmune sera. Moreover, this band disappears upon preabsorbtion of the antibody with the corresponding purified GST-hCLK2 protein but not upon preabsorbtion with other unrelated bacterially expressed proteins, including GST fusions. Also, the intensity of this band is drastically reduced in hclk-2-siRNA-treated cells as compared with controls. The serum from rabbit 2780 gave the strongest reaction and was used for immunoblot analyses throughout this study. Immunoblot Analysis—Cultured cells were trypsinized and pelleted and then resuspended in 5× volumes of extraction buffer (500 mm NaCl, 20 mm Tris, pH 8.0, 1% Nonidet P-40, 1 mm dithiothreitol, and protease inhibitors (Roche Applied Science)). The resuspended cells were submitted to five freeze-thaw cycles (frozen in liquid nitrogen and thawed at 37 °C). Cell debris were removed by centrifugation, and the quantity of protein was measured (Bio-Rad protein assay). 50 μg of protein were separated on 7.5 or 12% polyacrylamide gels and transferred to nitrocellulose. The membranes were preincubated in blocking solution (TBS-T plus 5% nonfat milk) at room temperature for 1 h and then incubated with the primary antibody at 4 °C overnight at the following concentrations: rabbit anti-hCLK2 antibody (1:500 to 1:1000), mouse anti-α-tubulin antibody (1:10000; Sigma), rabbit anti-actin antibody (1:500; Sigma), mouse anti-cytochrome c (1–2 μg/ml; Molecular Probes, Eugene, OR), and mouse anti-p300 (2 μg/ml; Upstate Biotechnology, Inc., Lake Placid, NY). After 3 × 15 min TBS-T washes, the membranes were incubated in blocking solution at room temperature for 2 h. The membranes were then incubated with donkey anti-rabbit IgG secondary antibody (1:3000; Jackson Immunoresearch Laboratories) or goat anti-mouse IgG (1:20000; Pierce) at room temperature for 1 h, followed by three 15-min TBS-T washes. Finally, the signal was detected by chemiluminescence (Amersham Biosciences). Immunostaining—Cells were transiently transfected with a plasmid, and 24 h later, they were seeded on coverslips. Forty-eight hours later, the coverslips were fixed in 4% paraformaldehyde/PBS for 10 min, permeabilized in acetone for 3 min, and then incubated at room temperature for 1 h with rabbit polyclonal anti-hCLK2 (2780, 2838, 1:100–1000), followed by biotinylated goat anti-rabbit or mouse IgG (1:5000) for 1 h. Finally, the cells were incubated with fluorescein-conjugated streptavidin (10 μg/ml) for 30 min and viewed under a Leitz fluorescence microscope. Similar results were obtained with 2780 and 2838 sera. The pattern observed was not detected by the preimmune sera or the secondary antibody alone. In addition, the observed pattern disappears upon preabsorbtion of the antibody with the corresponding purified GST-hCLK2 protein but not upon preabsorbtion with other unrelated bacterially expressed proteins, including GST fusions. Growth Rate Assay—Cells were seeded in six-well dishes at 1 × 105/well. At the times indicated, the cells were trypsinized and counted with a hemocytometer. Cell Death Assay—Cells were seeded at 1 × 105 in six-well dishes. The next day, the cells were treated by γ-ray (20 grays) and counted 72 h later. A series of different apoptosis-inducing agents was also investigated, and the cells were analyzed at various times following treatment (see Table II). Cell viability was measured by the trypan blue exclusion method, by counting with a hemocytometer.Table IICell death assaysTreatmentWorking concentrationTime of treatmenthEtoposide100 μM24Sodium azide15 μM48Menadione12 μM24Anisomycin2 μM16t-Butyl hydroperoxide40 μM48Staurosporine2 μM24All-trans-retinoic acid4 μM96Hydrogen peroxide0.5 μM24Juglone0.5 μM24Hydroxyurea0.6 mM96Tunicamycin5 μg/ml24 Open table in a new tab Measuring the Length of Telomeres—Genomic DNA from cultured cells was recovered by phenol-chloroform extraction and ethanol precipitation. 10 μg of DNA was digested by HinfI and RsaI (10 units/μg DNA) at 37 °C overnight. The completely digested DNA was separated on 0.7% agarose gel at 23 V for 24 h and transferred by capillary transfer to a positively charged nylon membrane (Amersham Biosciences) overnight. The telomere-specific sequence (5′-TTAGGGTTAGGGTTAGGG-3′) was used as a probe to detect telomeric repeats. The membrane was incubated in prehybridization solution (5× SSC, 5× Denhardt's solution, 0.1% SDS) for 1 h at 50 °C, followed by an overnight incubation in hybridization solution (5× SSC, 0.1% SDS, and 5′-32P-end-labeled probe) at 37 °C. The membrane was then washed in 3× SSC, 0.1% SDS at 42 °C for 3 × 10 min and exposed at room temperature overnight. Preparation of Subcellular Fractions—Subcellular fractionation was performed as described (18Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). From 1 to 10 × 107 cells were washed twice with ice-cold PBS and resuspended in buffer (0.25 m sucrose, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, protease inhibitors (Roche Applied Sciences)) at a concentration of 2 × 107 cells/ml. Cells were homogenized on ice (10–20 strokes at 1000 rpm; Potter-Elvehjem) until 95% of the cells were lysed based on trypan blue dye uptake. The samples were transferred to 1.5-ml Eppendorf centrifuge tubes (1 ml/tube) and centrifuged at 500 × g for 5 min to pellet the nuclei. The nuclear pellet was then resuspended in 0.5–2 ml of 1.6 m sucrose containing 50 mm Tris-HCl, pH 7.5, 25 mm KCl, 5 mm MgCl2. After underlayering with 1–2 ml of 2.0–2.3 m sucrose containing the same buffer and centrifugation at 150,000 × g for 60 min, the resulting nuclear pellets were resuspended in 0.1–0.3 ml of 1% Triton X-100-containing buffer (0.15 m NaCl, 10 mm Tris (pH 7.4), 5 mm EDTA, 1% Triton X-100). The supernatant resulting from the initial low speed centrifugation was subjected to centrifugation at 10,000 × g for 15 min at 4 °C to obtain the heavy membrane fraction (a pellet that should include mitochondria, lysosomes, Golgi, and rough endoplasmic reticulum). The supernatant was centrifuged for 60 min at 15,000 × g to obtain the light membrane fraction (a pellet that should include the smooth and rough endoplasmic reticulum) and the cytosolic fraction (supernatant). The heavy membrane and light membrane fractions were resuspended in 1% Triton-containing lysis buffer. An equal amount of protein (50 μg) from each fraction was analyzed by immunoblot. Growth Stimulation by Overexpression of hCLK2 in SK-HEP-1 Cells—To achieve high levels of hCLK2 expression in cultured cells, we used a retroviral vector expressing hCLK2 to infect a panel of cell lines (see "Experimental Procedures") and established stable cell lines, derived from pools of cells infected with the vector expressing hCLK2 or the empty vector control. A high level of hCLK2 expression was detected in all of the established cell lines (Fig. 1A and data not shown). In every case, the cells expressing hCLK2 did not show any morphological alterations compared with controls (data not shown). We found, however, that the growth rate of SK-HEP-1 (19Heffelfinger S.C. Hawkins H.H. Barrish J. Taylor L. Darlington G.J. In Vitro Cell Dev. Biol. 1992; 28A: 136-142Crossref PubMed Scopus (132) Google Scholar) cells overexpressing hCLK2 was increased over the control line (Fig. 2A), indicating that growth rate is sensitive to the level of hCLK2. We then used SK-HEP-1 cells for all subsequent characterization of the function of hCLK2. Other cell lines did not display obvious effects on growth, and their phenotype was not studied further (see "Experimental Procedures").Fig. 2The growth rate of SK-HEP-1 cells is affected by the level of expression of hCLK2.A, SK-HEP-1 cells overexpressing hCLK2 and control cells were plated at a density of 1 × 105/well in a six-well dish. At the indicated time (in days), cells were harvested, and the number of cells were counted using a hemocytometer. B, SK-HEP-1 cells were plated at a density of 1.0 × 105/well in a six-well dish and treated by siRNA or buffer the next day (day 1) at a density of about 1.5 × 105/well. Cell counts were done as above. For both panels, the means and S.E. of triplicate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reducing the Level of hCLK2 Expression Causes Reversible Growth Arrest—To investigate the consequences of a loss of function of hclk2, we used the siRNA technique (17Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8185) Google Scholar). SK-HEP-1 cells were treated with either 1) hclk2-specific siRNA, 2) siRNA for luciferase, a gene that is not normally found in human cells, or 3) the same volume of siRNA annealing buffer. The level of hCLK2 and the cell number were determined daily for several days following siRNA treatment (Figs. 1B and 2B). The immunoblots demonstrate that when the cells were treated with hclk2-specific siRNA, the level of hCLK2 was significantly decreased by day 2 and remained low until at least day 6. As expected, neither luciferase siRNA nor siRNA annealing buffer alone resulted in a decrease of the expression of hCLK2. In addition, the expression of actin was not affected by hclk2- specific siRNA, luciferase siRNA, or siRNA annealing buffer alone (Fig. 1B). hclk2 siRNA treatment dramatically slowed cellular growth rate, in contrast to treatment with luciferase siRNA, which had only a minor effect (Fig. 2B). The effect on growth rate lasted until day 7, after which time the cells appeared to recover from the treatment and resumed growth. No increase in cell death or other obvious changes were observed, indicating that the arrest was not the consequence of major damage to the cells. Treated cells were also sorted by fluorescence-activated cell sorting according to DNA content (data not shown). The arrested cells treated with hclk2 siRNA did not appear to have arrested in any particular phase of the cell cycle. Overexpression of hCLK2 Produces Hypersensitivity to Apoptosis Triggered by Oxidative Stress or DNA Replication Block—Prompted by the findings in the germ line of C. elegans, where clk-2 mutations affect the response to ionizing radiation and to DNA replication block induced by HU, we investigated the response of SK-HEP-1 cells overexpressing hCLK2 to 10 different agents capable of inducing apoptotic cell death as well as to HU and γ-rays. The cells overexpressing hCLK2 did not show any general increase in sensitivity to apoptotic stimuli but were specifically hypersensitive to two methods of increasing oxidative stress: menadione treatment, which leads to intracellular overproduction of superoxide (20Jamieson D.J. Rivers S.L. Stephen D.W. Microbiology. 1994; 140: 3277-3283Crossref PubMed Scopus (118) Google Scholar), and t-butyl hydroperoxide treatment, which leads to the production of the highly toxic hydroxyl radical (21Sano M. Kawabata H. Tomita I. Yoshioka H. Hu M.L. J. Toxicol. Environ. Health. 1994; 43: 339-350Crossref PubMed Scopus (6) Google Scholar) (Fig. 3A). The cells were also hypersensitive to the DNA synthesis inhibitor HU (Fig. 3A). To verify that the cell death observed was indeed apoptotic, we stained the cells using the TUNEL method (22Desjardins L.M. MacManus J.P. Exp. Cell Res. 1995; 216: 380-387Crossref PubMed Scopus (85) Google Scholar), which consists of in situ labeling of the 3′-OH ends of the cleaved DNA typical of apoptotic cells. A significant increase in the number of TUNEL-positive nuclei was observed in cells treated with the compounds that produced increased cell death compared with controls, namely menadione, t-butyl hydroperoxide, and hydroxyurea (Fig. 3B). We have also investigated the response of siRNA-treated SK-HEP-1 cells and found that the cells depleted for hCLK2 did not show any general increase in sensitivity to apoptotic stimuli (data not shown). It is unclear whether a reduction in hCLK2 levels has no effect on the sensitivity of the cells to the agents used or whether the arrest produced by siRNA treatment prevents the detection of any effect. Overexpression of hCLK2 Gradually Lengthens Telomeres—To investigate whether hclk2 affects telomere length in human cells, as it does in S. cerevisiae and in C. elegans, we determined the telomere length of SK-HEP-1 cells overexpressing hCLK2 and of SK-HEP-1 control cells by Southern blot analysis. We examined the telomere length at regular intervals during prolonged culturing (138 population doublings) (Fig. 4). The telomere length of the cells overexpressing hCLK2 gradually grew longer at an average rate of ∼15 bp/population doubling, whereas it remained absolutely stable in the control cells (Fig. 4). Additional population doublings do not appear to increase telomere length further (data not shown). hCLK2 Is Present in Most Compartments of the Cell—To determine the subcellular localization of hCLK2, we used immunocytochemistry to detect native and overexpressed hCLK2 in SK-HEP-1 cells. The level of native hCLK2 appeared to be too low to be detectable by this method with our antisera directed against hCLK2 (see "Experimental Procedures"). However, in cells overexpressing hCLK2, the signal appeared to be everywhere in the cell, filling both the cytoplasm and the nucleus (Fig. 5A). The same distribution was also observed in another overexpressing cell line HT-1080 (Fig. 5B). Controls included immunocytochemistry using the preimmune sera, the secondary antibody alone, and sera preabsorbed with a number of bacterial antigens. We determined the subcellular distribution of hCLK2 by immunocytochemistry following treatment with etoposide and menadione, two apoptotic triggering agents, which result in DNA replication inhibition and o
